M. Tuberculosis is an important human pathogen of serious and escalating morbidity and mortality. Our ability to develop new approaches to preventing and treating tuberculosis is hampered by our lack of knowledge of the intracellular biology of M. tuberculosis, including the composition of its phagosome and the interaction of the phagosome with other organelles of the host cell - the human mononuclear phagocyte. This proposal seeks to use cell biological, molecular biological, biochemical, and immunological approaches to characterize the composition of the phagosome - both in terms of host cell proteins and in terms of mycobacterial antigens - and to characterize the interaction of the phagosome with the host cell. In preliminary studies, we have used the cryosection immunogold technique to determine which known markers of the endosomal-lysosomal pathway are present on the M. tuberculosis phagosome. Based upon the results of these studies, we have hypothesized the M. tuberculosis arrest the maturation of its phagosome at an early endosome- like stage. This proposal will further explore this hypothesis, among other goals. Specific goals and aims of this proposal are: Goal I) Define the specific intracellular compartment in which M. tuberculosis resides in host mononuclear phagocytes. Aim a) Characterize the contribution of host cell molecules to the composition of the M. tuberculosis phagosome at various times after infection. Aim b) Characterize the interaction of the M. tuberculosis phagosome with endocytic and exocytic pathways. Aim c) Determine the effect of various cytokines and calcitriol on the composition of the mycobacterial phagosome and its interaction with other host cell organelles. Goal II) Characterize the contribution of M. tuberculosis molecules to the composition of its phagosome and determine if M. tuberculosis antigens are transported to extraphagosomal compartments. Aim d) Determine which M. tuberculosis proteins are induced or repressed during intracellular growth in human mononuclear phagocytes, and determine whether these responses mirror the mycobacterial response to environmental stresses (heat shock, osmotic shock) when growing extracellularly. Aim e) Determine which M. tuberculosis antigens are present on and in the phagosome at various times after infection. Aim f) Determine if M. tuberculosis antigens enter extraphagosomal host cell compartments. Data obtained from these studies will shed considerable light on the intracellular biology of M. tuberculosis, including mechanisms of virulence of M. tuberculosis, and on the host cell response to M. tuberculosis infection. By providing new insights into the biology of M. tuberculosis, these studies will hopefully lead to new strategies for the prevention and treatment of tuberculosis.